Proper regulation of signal transduction in cells is responsible for a variety of biological functions including normal cellular replication, growth, cell physiology and cell death. Any perturbations to normal signal transduction in cells can result in various disease states of the body and often disease states are a result of the involvement of more than one cell type and overall body physiology states. Specifically, in the case of cancer, this situation is especially intricate as there is involvement of many underlying inflammatory states of the human body. Diverse inflammatory conditions such as obesity, allergy, arthritis, and diabetes all play a huge role in how cancer progresses and how treatment may work. Therefore, creation of in vivo models that mimic complicated conditions such as cancer require animal models that have an active immune system. Without an active immune system, the dynamic cellular heterogeneity observed in cancer cannot be completely replicated. Furthermore, for clinical usefulness of such animal models of cancer, especially for prediction of the biology of each individual's cancer, organ invasion and cancer cell metastasis to other parts of the body, there should be a way of mimicking an individual patient's cancer in a very short time (before the start of chemotherapy) and predict cancer cell response to treatment.
For epithelial-based cancers, such as cancers of the breast, prostrate, lung, colon and pancreas, the need to focus therapy towards such metastasized tumors is of paramount importance. Invasive with distant metastasized stage IV carcinomas present a very low survival rate (seer.cancer.gov).
Metastatic cancer involves the detachment of aggressive malignant cells from the primary tumor into the bloodstream and/or lymphatic channels. Such circulating tumor cells (CTC) manage to reach distant organs where they develop secondary metastasis. Concordantly, the presence of these CTCs is associated with a poor prognosis (Balic M, Williams A, Lin H, Datar R, Cote R J. (2012). Circulating Tumor Cells: From Bench to Bedside. Annu Rev Med. 2012 Oct. 18.).
The treatment of patients with metastatic disease continues to be largely dependent on the information we obtain from the primary tumor in spite of frequent discordance between the biomarkers observed on primary tumors versus those observed on secondary tumors (Naoki Niikura, Jun Liu, Naoki Hayashi, Elizabeth A. Mittendorf, Yun Gong, Shana L. Palla, Yutaka Tokuda, Ana M. Gonzalez-Angulo, Gabriel N. Hortobagyi and Naoto T. Ueno (2011); Loss of Human Epidermal Growth Factor Receptor 2 (HER2) Expression in Metastatic Sites of HER2-Overexpressing Primary Breast Tumors. J Clin Oncol, 30:593-599; Dupont Jensen J, Laenkholm A V, Knoop A, Ewertz M, Bandaru R, Liu W, Hackl W, Barrett J C, Gardner H. (2011); PIK3CA mutations may be discordant between primary and corresponding metastatic disease in breast cancer. Clin Cancer Res. 17:667-77). As the circumstantial originators of secondary tumors and metastasis, understanding the biology of secondary tumors will add new perspectives in the individualized treatment of advanced cancer patients. In support of our hypothesis, the prognostic significance of CTCs has been demonstrated for several types of cancers (Cristofanilli M, Budd G T, Ellis M J, Stopeck A, Matera J, Miller M C, Reuben J M, Doyle G V, Allard W J, Terstappen L W, Hayes D F. (2004); Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 351:781-91; Moreno J G, Miller M C, Gross S, Allard W J, Gomella L G, Terstappen L W. (2005); Circulating tumor cells predict survival in patients with metastatic prostate cancer. Urology 65:713-8; Cohen S J, Punt C J, Iannotti N, Saidman B H, Sabbath K D, Gabrail N Y, Picus J, Morse M A, Mitchell E, Miller M C, Doyle G V, Tissing H, Terstappen L W, Meropol N J. (2009); Prognostic significance of circulating tumor cells in patients with metastatic colorectal cancer. Ann Oncol. 20:1223-9; Krebs M G, Sloane R, Priest L, Lancashire L, Hou J M, Greystoke A, Ward T H, Ferraldeschi R, Hughes A, Clack G, Ranson M, Dive C, Blackhall F H. (2011); Evaluation and prognostic significance of circulating tumor cells in patients with non-small-cell lung cancer. J Clin Oncol. 29:1556-63).
Molecular and genomic profiling of cancer cells has become the new trend in targeted therapy and oncology research. However, the relevance of molecular heterogeneity of the cancer cells and their constantly changing nature, the relevance of molecular signatures of the primary tumor as well as the CTCs is limited (Powell A A, Talasaz A H, Zhang H, Coram M A, Reddy A, et al. (2012) Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines. PLoS ONE 7: e33788.).
Molecular and genomic profiling of cancer cells has become important because it can provide targeted therapy for an individual's particular cancer. However, profiling of the primary tumor will not represent the molecular changes that have occurred in the metastatic CTC. What is required for the targeted treatment of metastatic secondary tumors is a way to profile the CTCs.
However there are very few CTCs in a patient's blood so it is very difficult to isolate and characterize the cells. Furthermore, isolating the few CTC in a patient's blood has limited applications, unless the cells can be propagated and examined Growing the CTC in tissue culture may be possible, but in vitro culture does not fully represent the cell characteristics, in particular their ability to invade normal tissues and form three-dimensional tumors, and to recruit growth factors and blood vessels.
The zebrafish, Danio rerio, a popular fresh water aquarium fish, is an important model organism and is being increasingly used in scientific research (Lieschke and Currie (2007) “Animal models of human disease: zebrafish swims into view.” Nature Reviews Genetics 8:353-367). In medicine, zebrafish has been extremely popular in the study of embryogenesis, cardiovascular research, neuronal development and retinal regeneration but recently it has been established as a great model for almost every kind of cancer as well (Stoletov and Klemke (2008) “Catch of the day: zebrafish as a human cancer model. Oncogene 27:4509-4520)”.
Zebrafish are responsive to carcinogenic chemicals and form neoplasms very similar to that seen in humans (Beckwith et al (2000) “Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio). Lab Invest. 80(3):379-385). It is also a fantastic model for cancer genetics (Stern and Zon (2003). “Cancer genetics and drug discovery in the zebrafish.” Nature Rev. Cancer 3: 533-539). The ease of genetic manipulations in zebrafish has aided its role in being an excellent model for understanding angiogenesis, apoptosis and metastasis (Serbedija et al (1999) “Zebrafish angiogenesis: a novel model for drug screening.”; Angiogenesis 3:353-359; Parng et al (2002) “Zebrafish: a preclinical model for drug screening.”; Assay Dev. Technol. 1:41-48; Marques et al (2009) “Metastatic behavior of primary human tumours in a zebrafish xenotransplantation model.” BMC Cancer 9:128).
Manipulations in zebrafish are performed at various stages of its growth, but 48 hours post fertilization (hpf) is frequently used and is one of the high priority stages for manipulations. The time and manpower required for the processing of many zebrafish embryos during large scale genetic, drug screening and toxicity studies, and cancer cell assays can often be the limiting factor for most laboratories. However, there are presently no commercially available multi-well microinjection systems for 48 hpf zebrafish embryos, primarily because of their elongated and odd shape.
Automated multi-well microinjection systems are well known in the field of cell biology wherein they are primarily used in intranuclear or intracytoplasmic injection of materials such as DNA, RNAi, proteins, or even other cells such as sperm. Automated systems enable a large number of microinjections with reproducible consistency and accuracy that is often hard to achieve manually.
Therefore what is needed to profile and characterize primary tumor cells and CTC is a method to establish and grow the tumor cells in vivo in an animal model. This could allow drug testing on the tumor cells and could provide targeted therapy to the tumor cells in the patient. Furthermore, what is needed in the art is a system that would enable efficient manipulation and injection of 48 hpf zebrafish embryos, for genetic, toxicity, drug, and cancer studies.